Bottom Line:
Here, we show that estrogen-induced, osteoanabolic effects were mediated via enhanced production of chondroitin sulfate-E (CS-E), which could act as an osteogenic stimulant in our cell-based system.Conversely, estrogen deficiency caused reduced expression of CS-E-synthesizing enzymes, including GalNAc4S-6ST, and led to decreased CS-E production in cultures of bone marrow cells derived from ovariectomized mice.Moreover, Galnac4s6st-deficient mice had abnormally low bone mass that resulted from impaired osteoblast differentiation.

ABSTRACTOsteoporosis is an age-related disorder of bone remodeling in which bone resorption outstrips bone matrix deposition. Although anticatabolic agents are frequently used as first-line therapies for osteoporosis, alternative anabolic strategies that can enhance anabolic, osteogenic potential are actively sought. Sex steroid hormones, particularly estrogens, are bidirectional regulators for bone homeostasis; therefore, estrogen-mediated events are important potential targets for such anabolic therapies. Here, we show that estrogen-induced, osteoanabolic effects were mediated via enhanced production of chondroitin sulfate-E (CS-E), which could act as an osteogenic stimulant in our cell-based system. Conversely, estrogen deficiency caused reduced expression of CS-E-synthesizing enzymes, including GalNAc4S-6ST, and led to decreased CS-E production in cultures of bone marrow cells derived from ovariectomized mice. Moreover, Galnac4s6st-deficient mice had abnormally low bone mass that resulted from impaired osteoblast differentiation. These results indicated that strategies aimed at boosting CS-E biosynthesis are promising alternative therapies for osteoporosis.

f4: Osteoblastic potential of BMSCs isolated from Galnac4s6st+/+ or Galnac4s6st−/− mice.(a,b) Expression of Akp2 mRNA in BMSCs cultured in a differentiation medium (DM) for 4 days in the presence or absence of estradiol (n = 3 cultures total, each from different litters). Data are represented as mean ± s.d. *, P < 0.05. (c,d) BMSCs were maintained in DM for 21 days and stained for ALP after 4 days of culture (c). Alizarin red staining was used to assess mineralized nodule formation (d). (e,f) Expression of Akp2 mRNA in 24-h cultures of BMSCs in the presence or absence of GAGs, CS-E or heparin (20 μg/ml each). (n = 3 cultures total, each from different litters). Data are presented as mean ± s.d. **, P < 0.01; ***, P < 0.001, Dunnett's test in f.

Mentions:
To examine whether the osteopenic/osteoporotic phenotypes of Galnac4s6st−/− mice were caused by defects in bone anabolism, the osteoblastic potential of BMSCs, derived either from WT or Galnac4s6st−/− mice, were assessed. As reported previously36, we confirmed that Galnac4s6st−/− BMSCs also produced CS completely devoid of E units (Table 3). Osteoblast differentiation and maturation constitute a highly ordered process that begins with ALP expression and ends with mineral deposition; therefore, isolated BMSCs were cultured in an osteogenic medium; on days 4 and 21, cells were stained for ALP and mineralized nodule formation, respectively. ALP expression was significantly lower in Galnac4s6st−/− BMSC cultures than in WT controls (Fig. 4a,c). Mineralized nodule formation in Galnac4s6st−/− BMSC cultures was also severely impaired (Fig. 4d). Notably, bath application of estradiol led to a significant increase in ALP expression and mineral deposition in WT BMSC cultures, but not in Galnac4s6st−/− BMSC cultures (Fig. 4a–d). We previously showed that exogenous addition of CS-E polysaccharides could increase ALP expression in the low-density MC3T3-E1 cultures24, and such a stimulatory effect was not compensated by heparin, another class of highly sulfated GAGs (Supplementary Fig. S3). Consistent with these findings, bath-applied CS-E, but not heparin, significantly augmented ALP expression even in initial 24-h cultures of Galnac4s6st−/− BMSCs (Fig. 4f), although the ALP level remained lower than that obtained from intact Galnac4s6st+/+BMSCs cultures (Fig. 4e,f). These data indicated that CS-E formed by GalNAc4S-6ST is essential for osteoblast differentiation and maturation, and that estrogens are potent inducers of CS-E-mediated osteoblastgenesis.

f4: Osteoblastic potential of BMSCs isolated from Galnac4s6st+/+ or Galnac4s6st−/− mice.(a,b) Expression of Akp2 mRNA in BMSCs cultured in a differentiation medium (DM) for 4 days in the presence or absence of estradiol (n = 3 cultures total, each from different litters). Data are represented as mean ± s.d. *, P < 0.05. (c,d) BMSCs were maintained in DM for 21 days and stained for ALP after 4 days of culture (c). Alizarin red staining was used to assess mineralized nodule formation (d). (e,f) Expression of Akp2 mRNA in 24-h cultures of BMSCs in the presence or absence of GAGs, CS-E or heparin (20 μg/ml each). (n = 3 cultures total, each from different litters). Data are presented as mean ± s.d. **, P < 0.01; ***, P < 0.001, Dunnett's test in f.

Mentions:
To examine whether the osteopenic/osteoporotic phenotypes of Galnac4s6st−/− mice were caused by defects in bone anabolism, the osteoblastic potential of BMSCs, derived either from WT or Galnac4s6st−/− mice, were assessed. As reported previously36, we confirmed that Galnac4s6st−/− BMSCs also produced CS completely devoid of E units (Table 3). Osteoblast differentiation and maturation constitute a highly ordered process that begins with ALP expression and ends with mineral deposition; therefore, isolated BMSCs were cultured in an osteogenic medium; on days 4 and 21, cells were stained for ALP and mineralized nodule formation, respectively. ALP expression was significantly lower in Galnac4s6st−/− BMSC cultures than in WT controls (Fig. 4a,c). Mineralized nodule formation in Galnac4s6st−/− BMSC cultures was also severely impaired (Fig. 4d). Notably, bath application of estradiol led to a significant increase in ALP expression and mineral deposition in WT BMSC cultures, but not in Galnac4s6st−/− BMSC cultures (Fig. 4a–d). We previously showed that exogenous addition of CS-E polysaccharides could increase ALP expression in the low-density MC3T3-E1 cultures24, and such a stimulatory effect was not compensated by heparin, another class of highly sulfated GAGs (Supplementary Fig. S3). Consistent with these findings, bath-applied CS-E, but not heparin, significantly augmented ALP expression even in initial 24-h cultures of Galnac4s6st−/− BMSCs (Fig. 4f), although the ALP level remained lower than that obtained from intact Galnac4s6st+/+BMSCs cultures (Fig. 4e,f). These data indicated that CS-E formed by GalNAc4S-6ST is essential for osteoblast differentiation and maturation, and that estrogens are potent inducers of CS-E-mediated osteoblastgenesis.

Bottom Line:
Here, we show that estrogen-induced, osteoanabolic effects were mediated via enhanced production of chondroitin sulfate-E (CS-E), which could act as an osteogenic stimulant in our cell-based system.Conversely, estrogen deficiency caused reduced expression of CS-E-synthesizing enzymes, including GalNAc4S-6ST, and led to decreased CS-E production in cultures of bone marrow cells derived from ovariectomized mice.Moreover, Galnac4s6st-deficient mice had abnormally low bone mass that resulted from impaired osteoblast differentiation.

ABSTRACTOsteoporosis is an age-related disorder of bone remodeling in which bone resorption outstrips bone matrix deposition. Although anticatabolic agents are frequently used as first-line therapies for osteoporosis, alternative anabolic strategies that can enhance anabolic, osteogenic potential are actively sought. Sex steroid hormones, particularly estrogens, are bidirectional regulators for bone homeostasis; therefore, estrogen-mediated events are important potential targets for such anabolic therapies. Here, we show that estrogen-induced, osteoanabolic effects were mediated via enhanced production of chondroitin sulfate-E (CS-E), which could act as an osteogenic stimulant in our cell-based system. Conversely, estrogen deficiency caused reduced expression of CS-E-synthesizing enzymes, including GalNAc4S-6ST, and led to decreased CS-E production in cultures of bone marrow cells derived from ovariectomized mice. Moreover, Galnac4s6st-deficient mice had abnormally low bone mass that resulted from impaired osteoblast differentiation. These results indicated that strategies aimed at boosting CS-E biosynthesis are promising alternative therapies for osteoporosis.